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Characteristics of the noradrenergic innervation of the left atrium in the chick (Gallus Gallus Domesticus, L.)
Comp 131o¢tk,m. Pttl~ud. It~=5 li~l 52(', pp 47 to 49 Iq,r!llzmon IJr~ss Pr:~ltcd I~I (atilt t'~itt,ll';
CHARACTERISTICS OF THE NORADRENERGIC INNERVATION OF" THE LEFT ATRIUM IN THE CHICK (GALLUS GALLUS DOMESTICUS, L.) T I RIN('I~ BENNFFI" AND T()RIU()RN M A I MF()RS
Department of Physk)logy. Medical School. Nottingham tlnixcrsit). Nottingham N(i7 2RD. l'ngland and Department of Toxicology. Astra Pharmaceuticals All, S-151 N5 S6dcrliilic. S~cden (Received 27 May 1975'i
I. The autonomic innervation of the left atrium ol the chick ~as studied. 2. The noradrenergic nerve supply caused a marked positive inotrop) ~ hen stmmlatcd. This eft'cot was enhanced by cocaine and phcnoxybenzamine, and was blocked b) propranolol. 3. The characteristics of the s.v.,,tem arc such that it might he useful for teaching purposes. Abstract
INTROI)I.I('I'ION THE INNERVATION Of the chick heart has been cxtcnsively documented from histochemical obscrvations (Bennett, 1974). However, in Htro stt, dics on the response of the avian heart to electrical stimulation of its nerve supply are less numerous: moreover, the investigations to date indicate certain oddities, particularly with regard to the noradrenergic mnervation, Thus, Bolton (1967) found that cocaine did not e n h a n c e the response of chick venlricular muscle to noradmnergic nerve stimulation: Bolton & Bowman (1969) also showcd lhat this drug had anomalous effects on the response of thc pcrfused chick heart to adrenaline. During the course of investigations into the processes of noradrenergic rc-mnervation of the denervatcd heart of the chick (Bennett & Mahnfors. 1974) observations were made thal arc pertinent to the above problems: they are reported here.
MATERIAI.S AND %IETHOI)S Two-week old White l.eghorn chicks were used throughout this stud).. Animals wcrc dccapilated t, ndcr light ether anaesthesia and the heart was remoxed: the left atrium was dissected free and suspcndcd betwccn parallel plait= hum wire electrodes m a jacketed, ItX) ml organ bath. The bath contained a saline solution (Ginsborg. 196()) that was gassed with 95",, 02.51':, CO2 and kept at 37 (.'. Contraclions of the tissue were recorded isometrically using a Statham transducer coupled to a Grass model 5 pol)graph. The left atrium is normally quiescent, since the pacemaker tissue is located in the right atrium: after preparations had equilibrated for 1 hr. they were driven electrically b) direct stimulation of the atrial musculature. This ~as achieved by application of pulses (411, 5 mscc duration. 6 V strength} to the platinum electrodes: such stimulation caused contraction of the atrium but no. or only minimal, excitation of the intramural cardiac nerves. When ncrve-mediated responscs were to be observed, the stimulus strength s~as increased from 6 to 60 V for 15 sec, thereby exciting nerxe fibres and muscle cells together. This mcthod permits observation of the motropic effects of nerve stimulation only (Blinks, 1966: Bolton. 1967). Drugs used in this stud), were: hyoscine h~drobromide, cocaine hydrochloride, phenoxybenzamme h~drochloride, and propranolol hydrochloride: concentrations 47
given m the text refer to thc.,,e salts. Numbers of experiinents are signilied b.', the ,,alue of it in the text. rot:st I.ls In the absence of drugs, stimulation ol + intramural nerve tibres causcd a biphasic response ta negative followed b.~ a positive motropy: see Bennett & Malmlots. 1974k or a negative inotrop.x alone (Fig. I). Thc killer effect ,aas blocked by hvoscinc [I x 10 " M, n = 10: Fig. 1) and thereafter, stimulation of intramural cardiac ncrvcs c,,okcd only a positive motropic effect (Fig 1). The dr, ration and amplitt,dc of this rcsponsc 'aas increased b) cocaine (1"5 x l0 " MI: in I0 cxpcfimcnts, the increase m dr, ration was 20 _*_ 5",, (mean _+ I S.t!.). and the mcrcasc m amplilude was 33 _- 7",, {mcan ~- I S.ILk Higher doscs of the drt, g 115 x 10 -5 M) caused greater increases in duration and amplitudc of the positi',e inotropic responsc to nclvc slinlulalion (Fig. 2i. l-lowc'~cr, m the presence of these high concentrations of cocaine, it was found that, ['ollm~ing a pcriod of ncrvc slimulation, the force of contraction of lhc atrium fell iramediately the stimulus xoltage v, as reduced (Fig. 2), furlhcrnlorc, the eft'cots of high concentrations of cocaine were onlx maximal ~ h c n the drug had been v,a s h c d from the bath (Fig. 21. "lhcsc obscrxations indicate that the high concentrations of cocaine ma 3 have been exerting a direct dcprcssanl cffccl on thc atrial nlusculaturc. The positive inotropic responses to intramural nerve librc stimu[ation were also enhanced b,, ph.cnox~bcn/amme {1.5 x 10 5 M, Jt = I(): Fig. 31: the mean increase in ampliludc of the response was 60 4- I(l",, while lhc duration was incrcascd b \ 62 4- 10",,. The posilive inotropic resf~onses to ncrxe slimu]alion ill the presence of phcnoxybcnzanlhlc x~crc .:tnhlgoni/cd b\ propranolo111.7 x It) " M. I:ig. 3). DIS.([ NNION The present invcstigation provides direct functional evidence that the left atrium in the chick receives an inhibitory and :.in excitatory mnervation. O n the basis
d,g
T. BIL~NI:II A',/D "]. MAI.MFORS
1
HYOS I
Fig. I. [notropic responses of the left atrium: the bars bclo~ the recording indicate periods of 15
sec during which thc intramural cardiac nerves were stimulated n~aximally. Note that. m the absence of drugs, such stimulation evoked only a negative inolropic cffcct. Addition of h;oscme (HYOS. I x 10-" M) to the bath caused a gradual increase in the force t~l contraction o f ' t h e atrium (duc to abolition of the effects of minimal cholmergic nerve stimulation), and rc,,calcd a pov,crtul positive inotropic effect in response to nerve stinmlation. Vertical calibration l.O g: horb, ontal calibration 5 rain.
of previous observations (Bennett. 1974). and the pharmacological findings reported here. it is likely that the inhibitory innervation is cholinergic while the excitatory innervation is noradrenergic. Our investigations indicate that the noradrenergic innervation has no unusual characteristics, except, perhaps, for the degree to which it is able to increase the force of contraction of the atrial muscle (see Fig. I). The present results are consonant with the ability of cocaine to inhibit the neuronal re-uptake of noradrenaline and thereby enhance the response to nerve stimulation (lversen, 1967). This does not preclude cocaine acting in a variety of other ways at the same time. Indeed, it was noticed that high doses of cocaine caused some depression of atrial contractility; it is possible that the failure of cocatine to enhance the response of ventricular muscle to noradrenergic nerve stimulation (Bolton, 1967), was associated with such a non-specific effect or. more likely', is related to the relatively sparse noradrenergic innervation of the yentricles (Bennett & Malmfors, 1970). It has been appreciated for some time that phenoxybenzamine has a variety of actions, causing blockade of alpha-receptors, increased release and decreased reuptake of noradrenaline by' noradrenergic nerves
(Iversen. 1967). ]'he actions of phenoxybcn/amine in the atrium providc a clear-cut demonstration of the ability of this drug to enhance the responses to noradrcncrgic ne,'ve stimulation. However. it is not possible to decide, from the present cxperiments, the relative contribt, lions that blockade of re-uptake and increased noradrenalme release made to the effects seen. It is kno,,vn thai cocaine is morc effective than phcnoxybcnzaminc in blocking noradrenaline uptake (Ivcrsen. 19671. and yet. in the present work. phenoxybenzanfine invariably caused a greater enhancement of the response to noradrcncrgic nerve stimulation than did cocaine. It is feasible that this difference is attribt, table to the ability of phenoxyben/amine to enhance noradrenaline release, possibly through its action on pres3naptic alpha receptors (cf. Starke et u/.. 19751. Previous investigations have indicated that the chick heart is more sensitive to noradrenaline than to adrenaline {Bolton. 1967; Bennett & Malmfors. 1974) and it has been noted that this differential scnsilivit~ is increased by noradrcnergic denervation (Bennett & Mahnfors. 1974). Nonetheless, the enhancemcnt of noradrencrgic nerve effects by phenoxybenTamine and their abolition by' propranolol confirms that
COC
coc
w J
l:ig. 2. Positive inotropic responses of the atrium in the presence of h.~oscinc: the gaps hct~een record-
rags represent 5 min periods. Addition ofcoc~,inc ~£'OC. I'5 x 10 " MI caused an increase in amplitude and duration of the responses. Further addition of cocaine (to Bring bath ctmccntration to 1"5 x lO s M) caused an enhancement that was only maximal after v.ashing lhe bath out (\VI: this indicatcs that this higher concentration of the drug may have been ha,,ing a depressant effect on the atrium (of. middle recording on the bottom row). Vertical calibration 0.5 g: horizontal calibration 5 nfin.
Avian cardiac innervaticm
49
l PROP
Fig. 3. Continuous recording of the positive inotropic effects of noradrencrgic nerve stimulation (ever,, 5 min) in the left atrium. Note that addition of phenox~benzamine to the organ bath (PHENOXY. 1.5 x 10-s M} caused an increase in the contractile force of the atrium and a marked enhancement of the responses to noradrenergic nerve stimulation. Thcsc effects were antagoni~,ed by propranolol {PROP. 1.7 x 10 " M). Vertical calibration 1.0 g: hori/ontal calibration 5 rain.
the receptors inw)lved are of the beta type. In the light o f these findings we w o u l d suggest that the isolated left a t r i u m of the chick might prove a useful p r e p a r a t i o n in the teaching o f cardiac muscle pharmacology, particularly in view o f its cheapness, , REFERENCE.'; BENNE'FT T. (1974) The peripheral and at, tonomic nervous systems. In Avian Bio/o.qy {Edited by Farxl:r D. S. & KXNG J. R.), Vol. IV, pp. I 77. Ac~ademic Press, New York. B~iyy~!l'r T. & MALMI-ORST. (1970)The adrcncrgic ncr,,ous system of the domestic fowl 4Gallu.s domesticus L.). Z. Zel!]'orsch. mikrosk. /bzat. 106, 22-50. BICNNHq T. & MAtM~ORS T. (1974) Regeneration of the noradrcnergic innervation of the cardiovascular ssstem of thc chick following treatment with 6-hydroxydopamine. J. Physiol.. Loml. 242. 517 532.
II I ' 5 2 h
I~
BI.INKSJ. R. (1966) Field stimulation as a means of effecting tbe graded release of autonomic transmitters in isolated heart muscle. J. Pharm. exp. Ther. t i l l 221-235. BOLT~')',;"F. B. ([967) Intramt, ral nerves in the ventricular myocardium of the domestic fo~.l and other animals. Br..I. Pharmac. 31, 253 268. BOI.ION T. B. & BOWMAN ~,V. C. (19691 Adrenoreceptors in the cardiovascuktr system of the domestic fowl. Eur. J. l~harmac. 5, 121 132. (}INSBORG B. L. (1960) Spontaneous acti,,ity in mu.~le fibres of the chick..I. Physiol. Loml. l,e'd). 707-717. I\'l:rsl:>: l.. 1_..(1967)The Uptake arid Stora~te ~?l Noradrenaline m Sympathetic .\"erre.s pp. 154 157. Cambridge University. Press. Sra~.Kll K.. [-yDo T. & Tat:l~l H. D. (1975) Prc- and postsynaptic components in cffect of drugs with zc adrenoceptot altinit 3. :\atlo'c. l,ond. 254. 440 441. Key II'ord huh'\ Atrium: noradrcncrgic inncrvation; cocaine: phcnox.,,bcnzaminc: chick : noradrcnaline; hcart.
CHARACTERISTICS OF THE NORADRENERGIC INNERVATION OF" THE LEFT ATRIUM IN THE CHICK (GALLUS GALLUS DOMESTICUS, L.) T I RIN('I~ BENNFFI" AND T()RIU()RN M A I MF()RS
Department of Physk)logy. Medical School. Nottingham tlnixcrsit). Nottingham N(i7 2RD. l'ngland and Department of Toxicology. Astra Pharmaceuticals All, S-151 N5 S6dcrliilic. S~cden (Received 27 May 1975'i
I. The autonomic innervation of the left atrium ol the chick ~as studied. 2. The noradrenergic nerve supply caused a marked positive inotrop) ~ hen stmmlatcd. This eft'cot was enhanced by cocaine and phcnoxybenzamine, and was blocked b) propranolol. 3. The characteristics of the s.v.,,tem arc such that it might he useful for teaching purposes. Abstract
INTROI)I.I('I'ION THE INNERVATION Of the chick heart has been cxtcnsively documented from histochemical obscrvations (Bennett, 1974). However, in Htro stt, dics on the response of the avian heart to electrical stimulation of its nerve supply are less numerous: moreover, the investigations to date indicate certain oddities, particularly with regard to the noradrenergic mnervation, Thus, Bolton (1967) found that cocaine did not e n h a n c e the response of chick venlricular muscle to noradmnergic nerve stimulation: Bolton & Bowman (1969) also showcd lhat this drug had anomalous effects on the response of thc pcrfused chick heart to adrenaline. During the course of investigations into the processes of noradrenergic rc-mnervation of the denervatcd heart of the chick (Bennett & Mahnfors. 1974) observations were made thal arc pertinent to the above problems: they are reported here.
MATERIAI.S AND %IETHOI)S Two-week old White l.eghorn chicks were used throughout this stud).. Animals wcrc dccapilated t, ndcr light ether anaesthesia and the heart was remoxed: the left atrium was dissected free and suspcndcd betwccn parallel plait= hum wire electrodes m a jacketed, ItX) ml organ bath. The bath contained a saline solution (Ginsborg. 196()) that was gassed with 95",, 02.51':, CO2 and kept at 37 (.'. Contraclions of the tissue were recorded isometrically using a Statham transducer coupled to a Grass model 5 pol)graph. The left atrium is normally quiescent, since the pacemaker tissue is located in the right atrium: after preparations had equilibrated for 1 hr. they were driven electrically b) direct stimulation of the atrial musculature. This ~as achieved by application of pulses (411, 5 mscc duration. 6 V strength} to the platinum electrodes: such stimulation caused contraction of the atrium but no. or only minimal, excitation of the intramural cardiac nerves. When ncrve-mediated responscs were to be observed, the stimulus strength s~as increased from 6 to 60 V for 15 sec, thereby exciting nerxe fibres and muscle cells together. This mcthod permits observation of the motropic effects of nerve stimulation only (Blinks, 1966: Bolton. 1967). Drugs used in this stud), were: hyoscine h~drobromide, cocaine hydrochloride, phenoxybenzamme h~drochloride, and propranolol hydrochloride: concentrations 47
given m the text refer to thc.,,e salts. Numbers of experiinents are signilied b.', the ,,alue of it in the text. rot:st I.ls In the absence of drugs, stimulation ol + intramural nerve tibres causcd a biphasic response ta negative followed b.~ a positive motropy: see Bennett & Malmlots. 1974k or a negative inotrop.x alone (Fig. I). Thc killer effect ,aas blocked by hvoscinc [I x 10 " M, n = 10: Fig. 1) and thereafter, stimulation of intramural cardiac ncrvcs c,,okcd only a positive motropic effect (Fig 1). The dr, ration and amplitt,dc of this rcsponsc 'aas increased b) cocaine (1"5 x l0 " MI: in I0 cxpcfimcnts, the increase m dr, ration was 20 _*_ 5",, (mean _+ I S.t!.). and the mcrcasc m amplilude was 33 _- 7",, {mcan ~- I S.ILk Higher doscs of the drt, g 115 x 10 -5 M) caused greater increases in duration and amplitudc of the positi',e inotropic responsc to nclvc slinlulalion (Fig. 2i. l-lowc'~cr, m the presence of these high concentrations of cocaine, it was found that, ['ollm~ing a pcriod of ncrvc slimulation, the force of contraction of lhc atrium fell iramediately the stimulus xoltage v, as reduced (Fig. 2), furlhcrnlorc, the eft'cots of high concentrations of cocaine were onlx maximal ~ h c n the drug had been v,a s h c d from the bath (Fig. 21. "lhcsc obscrxations indicate that the high concentrations of cocaine ma 3 have been exerting a direct dcprcssanl cffccl on thc atrial nlusculaturc. The positive inotropic responses to intramural nerve librc stimu[ation were also enhanced b,, ph.cnox~bcn/amme {1.5 x 10 5 M, Jt = I(): Fig. 31: the mean increase in ampliludc of the response was 60 4- I(l",, while lhc duration was incrcascd b \ 62 4- 10",,. The posilive inotropic resf~onses to ncrxe slimu]alion ill the presence of phcnoxybcnzanlhlc x~crc .:tnhlgoni/cd b\ propranolo111.7 x It) " M. I:ig. 3). DIS.([ NNION The present invcstigation provides direct functional evidence that the left atrium in the chick receives an inhibitory and :.in excitatory mnervation. O n the basis
d,g
T. BIL~NI:II A',/D "]. MAI.MFORS
1
HYOS I
Fig. I. [notropic responses of the left atrium: the bars bclo~ the recording indicate periods of 15
sec during which thc intramural cardiac nerves were stimulated n~aximally. Note that. m the absence of drugs, such stimulation evoked only a negative inolropic cffcct. Addition of h;oscme (HYOS. I x 10-" M) to the bath caused a gradual increase in the force t~l contraction o f ' t h e atrium (duc to abolition of the effects of minimal cholmergic nerve stimulation), and rc,,calcd a pov,crtul positive inotropic effect in response to nerve stinmlation. Vertical calibration l.O g: horb, ontal calibration 5 rain.
of previous observations (Bennett. 1974). and the pharmacological findings reported here. it is likely that the inhibitory innervation is cholinergic while the excitatory innervation is noradrenergic. Our investigations indicate that the noradrenergic innervation has no unusual characteristics, except, perhaps, for the degree to which it is able to increase the force of contraction of the atrial muscle (see Fig. I). The present results are consonant with the ability of cocaine to inhibit the neuronal re-uptake of noradrenaline and thereby enhance the response to nerve stimulation (lversen, 1967). This does not preclude cocaine acting in a variety of other ways at the same time. Indeed, it was noticed that high doses of cocaine caused some depression of atrial contractility; it is possible that the failure of cocatine to enhance the response of ventricular muscle to noradrenergic nerve stimulation (Bolton, 1967), was associated with such a non-specific effect or. more likely', is related to the relatively sparse noradrenergic innervation of the yentricles (Bennett & Malmfors, 1970). It has been appreciated for some time that phenoxybenzamine has a variety of actions, causing blockade of alpha-receptors, increased release and decreased reuptake of noradrenaline by' noradrenergic nerves
(Iversen. 1967). ]'he actions of phenoxybcn/amine in the atrium providc a clear-cut demonstration of the ability of this drug to enhance the responses to noradrcncrgic ne,'ve stimulation. However. it is not possible to decide, from the present cxperiments, the relative contribt, lions that blockade of re-uptake and increased noradrenalme release made to the effects seen. It is kno,,vn thai cocaine is morc effective than phcnoxybcnzaminc in blocking noradrenaline uptake (Ivcrsen. 19671. and yet. in the present work. phenoxybenzanfine invariably caused a greater enhancement of the response to noradrcncrgic nerve stimulation than did cocaine. It is feasible that this difference is attribt, table to the ability of phenoxyben/amine to enhance noradrenaline release, possibly through its action on pres3naptic alpha receptors (cf. Starke et u/.. 19751. Previous investigations have indicated that the chick heart is more sensitive to noradrenaline than to adrenaline {Bolton. 1967; Bennett & Malmfors. 1974) and it has been noted that this differential scnsilivit~ is increased by noradrcnergic denervation (Bennett & Mahnfors. 1974). Nonetheless, the enhancemcnt of noradrencrgic nerve effects by phenoxybenTamine and their abolition by' propranolol confirms that
COC
coc
w J
l:ig. 2. Positive inotropic responses of the atrium in the presence of h.~oscinc: the gaps hct~een record-
rags represent 5 min periods. Addition ofcoc~,inc ~£'OC. I'5 x 10 " MI caused an increase in amplitude and duration of the responses. Further addition of cocaine (to Bring bath ctmccntration to 1"5 x lO s M) caused an enhancement that was only maximal after v.ashing lhe bath out (\VI: this indicatcs that this higher concentration of the drug may have been ha,,ing a depressant effect on the atrium (of. middle recording on the bottom row). Vertical calibration 0.5 g: horizontal calibration 5 nfin.
Avian cardiac innervaticm
49
l PROP
Fig. 3. Continuous recording of the positive inotropic effects of noradrencrgic nerve stimulation (ever,, 5 min) in the left atrium. Note that addition of phenox~benzamine to the organ bath (PHENOXY. 1.5 x 10-s M} caused an increase in the contractile force of the atrium and a marked enhancement of the responses to noradrenergic nerve stimulation. Thcsc effects were antagoni~,ed by propranolol {PROP. 1.7 x 10 " M). Vertical calibration 1.0 g: hori/ontal calibration 5 rain.
the receptors inw)lved are of the beta type. In the light o f these findings we w o u l d suggest that the isolated left a t r i u m of the chick might prove a useful p r e p a r a t i o n in the teaching o f cardiac muscle pharmacology, particularly in view o f its cheapness, , REFERENCE.'; BENNE'FT T. (1974) The peripheral and at, tonomic nervous systems. In Avian Bio/o.qy {Edited by Farxl:r D. S. & KXNG J. R.), Vol. IV, pp. I 77. Ac~ademic Press, New York. B~iyy~!l'r T. & MALMI-ORST. (1970)The adrcncrgic ncr,,ous system of the domestic fowl 4Gallu.s domesticus L.). Z. Zel!]'orsch. mikrosk. /bzat. 106, 22-50. BICNNHq T. & MAtM~ORS T. (1974) Regeneration of the noradrcnergic innervation of the cardiovascular ssstem of thc chick following treatment with 6-hydroxydopamine. J. Physiol.. Loml. 242. 517 532.
II I ' 5 2 h
I~
BI.INKSJ. R. (1966) Field stimulation as a means of effecting tbe graded release of autonomic transmitters in isolated heart muscle. J. Pharm. exp. Ther. t i l l 221-235. BOLT~')',;"F. B. ([967) Intramt, ral nerves in the ventricular myocardium of the domestic fo~.l and other animals. Br..I. Pharmac. 31, 253 268. BOI.ION T. B. & BOWMAN ~,V. C. (19691 Adrenoreceptors in the cardiovascuktr system of the domestic fowl. Eur. J. l~harmac. 5, 121 132. (}INSBORG B. L. (1960) Spontaneous acti,,ity in mu.~le fibres of the chick..I. Physiol. Loml. l,e'd). 707-717. I\'l:rsl:>: l.. 1_..(1967)The Uptake arid Stora~te ~?l Noradrenaline m Sympathetic .\"erre.s pp. 154 157. Cambridge University. Press. Sra~.Kll K.. [-yDo T. & Tat:l~l H. D. (1975) Prc- and postsynaptic components in cffect of drugs with zc adrenoceptot altinit 3. :\atlo'c. l,ond. 254. 440 441. Key II'ord huh'\ Atrium: noradrcncrgic inncrvation; cocaine: phcnox.,,bcnzaminc: chick : noradrcnaline; hcart.